The purpose of this study is to detect beefy meaty peptide (BMP) in beef hydrolyzate. The synthesized BMP is used as a standard sample in the study. High performance liquid chromatography (HPLC)/ion trap electrospray ionization mass spectrometry (ESI-MS) with UV detection was applied in qualitative analysis of the peptides. Six beef protein enzymatic hydrolyzate samples were separated on a Surveyor HPLC system through a SUPELCO Discovery® C18 analytical column (5 μm, 15 cm×2.1 mm i.d.). The column was eluted at a flow rate of 0.2 mL/min in a linear gradient elution mode of acetronitrile-water solution with 0.1% trifluoroacetic acid. The concentration of acetronitrile was increased from 5% to 50% in 40 minutes. A Finnigan LCQ Advantage MAX instrument was used as detector to analyze with ESI-MS and ESI-MS/MS in positive mode. Among the six samples of beef protein enzymatic hydrolysate, the BMP is detected and confirmed in sample No.4 with a higher intensity of characteristic peak and is further investigated by ESI-MS/MS. As a result, BMP exists in sample No.4. The study proves that HPLC-ESI-MS/MS is a simple, rapid, sensitive method to analyze target peptides from complex polypeptides.